Viral heart disease (myocarditis and dilated cardiomyopathy (DCM)) are important causes of morbidity and mortality. The role of the innate immune system in these diseases remains poorly understood but the initial anti-viral response by the host is known to be mediated by Toll-like receptors (TLRs). Double stranded RNA is the primary ligand for TLR3 and we have shown that the integrity of the TLR3 signaling pathway is involved in protecting against enterovirus-induced myocardial damage. Activation of this pathway triggers production of type I interferon (IFN), which orchestrates both the rapid response and long-term resistance to viral infection. Although it has been suggested that the genetic composition of the host is likely to play a crucial role in determining susceptibility to viral heart disease, to date no susceptibility genes have been identified. We have identified several nonsynonymous substitutions in the TLR3 and TRIF genes in patients with viral myocarditis or DCM: preliminary characterization suggests that they reduce TLR3 signaling. Our hypothesis is that the innate immune system is critical for mediating the host immune response against viral infection of the heart and that defects result in increased pathology and worse outcomes in patients. To test this hypothesis we are proposing three Specific Aims: Specific Aim 1: Determine the functional significance of TLR3 and TRIF mutations/polymorphisms using in vitro (cell culture) assays of TLR signaling. To test whether polymorphisms or mutations in TLR3 or TRIF affect normal protein function and/or increase viral replication, we will express mutant forms of the gene in cell culture systems and then induce signaling through the use of ligands or virus infection. The effect on function will be determined by investigating interferon production, while the effect on viral replication will be measured by real time PCR. In addition, we will determine whether the TRIF variants act in dominant negative manner, and whether variants alter protein localization, turnover, as well as TLR3 dimerization and glycosylation. Specific Aim 2: Investigate the role of the cardiac innate immune response in protecting mice against cardiotropic virus infection. We will investigate whether the expression of mutated forms of TLR3 in the myocardium leads to increased cardiac pathology and/or decreased survival following infection with EMCV. PUBLIC HEALTH RELEVANCE: Many viruses can infect the heart, leading to disease, but in most people these viruses do not cause serious disease. It has been suggested that changes in genes may affect how an individual deals with a virus infection. In this project we will look at changes in the immune system and determine whether these could make a person more likely to develop heart disease following virus infection.